Intranasal HSV-1 Infection Drives Region-Specific Interferon-Dominant Microglial Remodeling

Background and Objectives Herpes simplex virus type 1 (HSV-1) is a neurotropic pathogen capable of invading the central nervous system (CNS) and increasingly associated with chronic neuroinflammation, cognitive impairment, and neurodegenerative disease. While microglia orchestrate the initial immune response to HSV-1, the molecular mechanisms that regulate their sustained neuroinflammatory activity in vivo remain poorly understood. Methods To define the transcriptional and epigenetic mechanisms that shape microglial responses during acute HSV-1 infection in vivo, we have, for the first time, integrated single-nucleus RNA sequencing, chromatin accessibility profiling, and spatial transcriptomics in a physiologically relevant intranasal HSV-1 infection model. Results Single-cell multiome analysis of CD11b⁺ nuclei identified transcriptionally and epigenetically distinct microglial and macrophage populations. HSV-1 infection redistributed monocyte-lineage states, with a marked overrepresentation of interferon (IFN)-responsive microglia and macrophage-associated populations. These states exhibited amplification of STAT1/2-, IRF1-, and CEBPB-centered regulons, distinguishing IFN-responsive microglia from macrophage-enriched populations rather than reflecting uniform activation. Homeostatic microglial gene signatures ( e.g., ApoE, Cst3 ) were reduced in response to HSV-1 infection. Spatial transcriptomics localized HSV-1 antigen to discrete brainstem regions, which were enriched for predicted STAT-, IRF-, and CEBPB-regulated targets identified through single-nuclei analysis. Discussion Using a multiomic framework, we demonstrate that HSV-1 infection drives transcriptional and epigenetic remodeling of microglial populations, characterized by a dominance of IFN-responsive states and a loss of homeostatic signatures. These findings provide mechanistic insight into how localized viral infection can reprogram microglial regulatory landscapes to maintain persistent HSV-1–associated neuroinflammation, contributing to long-term neurological vulnerability and neurodegenerative disease risk.